Potency of peptide + polyIC-LC +/- montanide vs CpG and mRNA/LNP for vaccination in C57Bl/6 mice.
Experiments here were planned, done, and reported by Jess Alley and Maria Sambade, with assistance from Blake Inabinett
In a prior experiment, we tested multiple vaccine formulations for capacity to elicit T cell responses in C57Bl/6 mice using a MHC class I-restricted murine cytomegalovirus epitope (M57) and an ELISPOT readout for interferon gamma production. We found that CpG (a TLR9 agonist) was the most potent peptide vaccine adjuvant tested, and that mRNA LNP vaccination was as potent as peptide + CpG vaccination. PolyIC (a TLR3 agonist) was less effective than CpG in our study. That result was less useful than we’d like, however, because the common form of polyIC used in human clinical trials for peptide-based cancer vaccines is augmented by the addition of poly-lysine (polyIC-LC). Some trials have also used polyIC-LC plus montanide ISA 51 (a water-in-oil emulsion adjuvant comprising mineral oil and a mannide monooleate surfactant mixed with the antigen peptide).
We are interested in the performance of vaccine formulations as therapeutic tumor antigen vaccines. To this end, we repeated a subset of our prior experiment adding in peptide + polyIC-LC +/- montanide ISA 51 vaccination as treatment groups (Figure 1).
As in our prior experiment, we gave a priming vaccine dose on day 0, a single boost dose on day 14, and then analyzed splenocytes on day 21 for antigen-specific reactivity by interferon gamma ELISPOT. We chose the polyIC-LC dose based on published work, where 50 µg has been routinely administered intramuscularly (Belnoue et al., 2016; Derouazi et al., 2015; Sultan et al., 2020; Tran et al., 2020; Weng et al., 2023; Zhu et al., 2007). We administered polyIC through two injections per time point in the current experiment (as opposed to a single injection per time point in previous experiments) to enable a direct comparison with polyIC-LC, which must be administered through two injections per time point due to the concentration at which this adjuvant is provided and limits on volumes administered intramuscularly in mice.
We also determined the montanide (ISA 51) dose based on published work. Although identification of actual ISA 51 doses used in prior peptide vaccination studies is challenging, a dose of 25-100 µL of ISA 51 mixed 1:1 (v/v) with some other diluent (PBS, peptide suspension, or other adjuvant solution) seems to be common in mice, with 50 µL ISA 51 (prior to 1:1 mixing) being the most frequently used dose (Beebe et al., 2008; Chawla et al., 2019; Chu et al., 2022; Gabri et al., 2006; Ghorbani et al., 2005; Rosenthal et al., 2017; Varypataki et al., 2016). In this experiment, we delivered a total of 50 µL ISA 51 (prior to diluting 1:1) at each vaccine time point. In both humans and mice, this adjuvant is commonly administered subcutaneously or intramuscularly (van Doorn et al., 2015). To be consistent with recent clinical trials (Slingluff et al., 2021), we administered ISA 151 subcutaneously after mixing it 1:1 with peptide suspended in poly-ICLC as per manufacturer recommendations. The preparation of this adjuvant is critical to its success, with the syringe-extrusion/ two-syringe technique recommended over vortexing to mix (Koh et al., 2006; van Doorn et al., 2015). In the current study, we used the syringe-extrusion/two-syringe mixing protocol that was obtained from Seppic, the manufacturer.
In this experiment, peptide + CpG and mRNA vaccination gave similar results (Figure 2). Although polyIC-LC may have boosted responses relative to polyIC, there was high variance in the polyIC-LC group, and the difference between polyIC and polyIC-LC was not statistically significant in this small study.
Going forward, we will test more vaccine formulations (more adjuvants and DC vaccines next, hopefully DNA vaccines after that), dose titrations of key formulations, different vaccine schedules (varying prime and boost timing and number of injections), and importantly, we will evaluate vaccination against a set of validated neoantigens (rather than the M57 antigen alone) in tumor-bearing mice. Should these results bear out in future studies, we will pursue peptide + CpG and/or mRNA/LNP vaccination for clinical translation.
Many thanks to the Jamie Leandro Foundation (a non-profit foundation dedicated to making therapeutic cancer vaccines available to patients) as well as Natera for funding this work and supporting making the results available on this platform.
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